Premature and critically ill newborns are among the most vulnerable of populations, requiring intensive nursing care characterized by multiple procedures, many of which are painful. Recent studies in both premature human newborns and neonatal rats provide evidence suggesting that the nervous system is more sensitive to painful stimuli during this time of unparalleled neurosensory plasticity. Given that repetitive nonpainful events in neonatal rats have been shown to affect neuroendocrine and immune responses to stress in mature animals, it is possible that repeated painful stressors might lead to even more profound effects in the mature animal. The overall objective of the funded study is to explore in the mature animal the biobehavioral consequences of repetitive neonatal pain. Fischer 344 rats exposed to either paw needle prick, paw touch or left unperturbed in their first 8 postnatal days and are assessed at maturity for responses to abdominal surgery or swim stress. Dependent variables include: (1) immune outcomes: natural killer (NK) function in vivo, the lung retention of syngeneic NK-sensitive MADB106 tumor cells, and ex-vivo, whole blood NK cytotoxicity against the standard YAC-1 target, and plasma interleukin-6; (2) neuroendocrine outcomes: plasma corticosterone and beta-endorphin; and (3) behavioral outcomes such as open field testing. The proposed expansion of the project adds: (1) maternal-pup interactions during the first postnatal week, as these relationships have been shown to play a role in the development of stress reactivity; (2) ex-vivo assessment of lung vascular NK cell activity against MADB106 tumor cells to reflect the ability of local NK cells to resist an organ-specific metastasis; (3) counts of both whole blood and lung vascular NK cells, a necessity for interpreting the lung NK assay, and a means by which to assess for changes in activity per NK cell or changes in NK cell number per ml whole blood to account for observed differences in whole blood NK activity; and (4) elevated plus-maze testing to provide an additional and possibly more sensitive measure of anxiety. These additional outcomes require no additional animals, yet have the potential to substantially increase the findings produced by this study as well as augment their interpretability. In sum, these additional outcomes strengthen the potential to demonstrate negative physiologic and behavioral sequelae of repeated painful experiences during neonatal development. [unreadable] [unreadable]